Abstract

Osteoclasts, together with osteoblasts, control the amount of bone tissue and regulate bone remodeling. Osteoclast differentiation is an important factor related to the pathogenesis of bone-loss related diseases. Reactive oxygen species (ROS) acts as a signal mediator in osteoclast differentiation. Simvastatin, which inhibits 3-hydroxy-3-methylglutaryl coenzyme A, is a hypolipidemic drug which is known to affect bone metabolism and suppresses osteoclastogenesis induced by receptor activator of nuclear factor-κB ligand (RANKL). In this study, we analyzed whether simvastatin can inhibit RANKL-induced osteoclastogenesis through suppression of the subsequently formed ROS and investigated whether simvastatin can inhibit H2O2-induced signaling pathways in osteoclast differentiation. We found that simvastatin decreased expression of tartrate-resistant acid phosphatase (TRAP), a genetic marker of osteoclast differentiation, and inhibited intracellular ROS generation in RAW 264.7 cell lines. ROS generation activated NF-κB, protein kinases B (AKT), mitogen-activated protein kinases signaling pathways such as c-JUN N-terminal kinases, p38 MAP kinases as well as extracellular signal-regulated kinase. Simvastatin was found to suppress these H2O2-induced signaling pathways in osteoclastogenesis. Together, these results indicate that simvastatin acts as an osteoclastogenesis inhibitor through suppression of ROS-mediated signaling pathways. This indicates that simvastatin has potential usefulness for osteoporosis and pathological bone resorption.

Introduction

Osteoclasts are known to participate in bone remodeling by removing the bone's mineralized matrix resulting in bone resorption. It has been well documented that excessive osteoclast formation is the cause of pathological bone diseases such as rheumatoid arthritis and osteoporosis (Boyle et al., 2003). Osteoclast differentiation of precursors can be induced by the presence of both receptor activator of NF-κB ligand (RANKL), a key factor which controls function and survival of mature osteoclasts (Yasuda et al., 1998; Suda et al., 1999; Teitelbaum and Ross, 2003) and macrophage-colony stimulating factor (M-CSF), which is produced by osteoblasts or T cells (Lacey et al., 1998; Fata et al., 2000; Teitelbaum, 2000). It is well known that the binding of RANKL to its receptor, receptor activator of nuclear factor κB (RANK), begins the induction of TNF receptor-associated factors (TRAFs) and activates multiple downstream signaling pathways such as NF-κB, protein kinases B (AKT), c-JUN n-terminal kinases (JNK), p38 MAP kinases (p38) and extracellular signal-regulated kinase (ERK) (Lee et al., 2001; Lee et al., 2003; Kwak et al., 2008).

Various growth factors and cytokines such as TNF-α have been shown to bind to receptors and generate reactive oxygen species (ROS) thus increasing intracellular ROS levels in various cells (Thannickal and Fanburg, 2000). At high concentrations, ROS oxidation can result in the inflammatory response, aging, apoptosis, and cancer. However, low levels of ROS may play a role as a secondary messenger in various signaling pathways (Sundaresan et al., 1995; Bae et al., 1997; Forman et al., 2004). Osteoclast lineages are very susceptible to oxidative stress, as osteoclasts are produced by increased-generation of intracellular ROS (Garrett et al., 1990; Steinbeck et al., 1994) and osteoclasts are activated by ROS (Garrett et al., 1990; Fraser et al., 1996). RANKL-induced intracellular ROS production serves to regulate the RANKL signaling pathways required for osteoclast differentiation and act as an upstream component of signaling pathways that mediate osteoclast activation and survival (Ha et al., 2004; Lee et al., 2005). Such signaling pathways also induce bone resorption by mature osteoclasts (Armstrong et al., 2002).

In this study, we investigated the effect of simvastatin on osteoclast differentiation and we found that simvastatin acts as a RANKL-mediated osteoclastogenesis inhibitor. This inhibitory effect appears to be due to suppression of ROS generation and simvastatin also blocked RANKL-induced signaling pathways (MAPKs, AKT and IκBα) which were required for osteoclast differentiation. It was showed that simvastatin inhibits osteoclast differentiation and osteoclast activity by decreasing the generation of intracellular ROS.

Results

Simvastatin inhibited RANKL-induced osteoclast differentiation

As a control, RANKL-induced osteoclast differentiation was tested in order to evaluate the effect of simvastatin on RANKL-induced osteoclast differentiation. RAW 264.7 cells were allowed to differentiate to osteoclast in the presence of RANKL and M-CSF for 7 days. Upon TRAP staining, the number of TRAP stained cells, indicating degree of osteoclast differentiation, increased in a time-dependent manner for 7 days. This is consistent with results from RT-PCR and real-time PCR analysis which showed a time-dependent increase of TRAP gene expression. In order to ensure that the added simvastatin would not have a cytotoxic effect, an MTT assay was conducted. The MTT assay showed no cytotoxic effect of simvastatin on cell within the used range in this study (<50 nM, data not shown). After verifying the validity of culture condition for osteoclast differentiation, simvastatin was added to RAW 264.7 cells at varying concentrations during RANKL-mediated osteoclast differentiation. As shown in Figure 1A, simvastatin inhibited RANKL-induced osteoclast differentiation. This is shown by a dose-dependent decrease in the formation of TRAP positive cells and simvastatin inhibited TRAP+ MNC generation by 73.7 ± 8.1 and 32.3 ± 8.0 at 30 and 50 nM concentrations, respectively (Figure 1B). Also, there were by a dose-dependent down-regulation of TRAP gene expression in RT-PCR (data not shown) and real-time PCR analysis (Figure 1C).

To investigate whether ROS production was induced during RANKL-stimulated osteoclast differentiation, intracellular ROS levels were analyzed with a cell-permeable, oxidation-sensitive dye DCFH-DA. ROS oxidation of this dye was detected by using laser-scanning confocal microscopy. RANKL and M-CSF stimulation was showed an increase in the intensity of DCF fluorescence indicating oxidation by H2O2 in RAW 264.7 cells (Figure 2A). This dose-dependent ROS production was also indicated by quantification analysis with Amplex Red reagent which reacted with H2O2 to produce a red-fluorescent oxidation product (Figure 2B).

Production of ROS following RANKL stimulation and effects of H2O2 on p-IκBα, p-AKT, p-MAPKs signals activation in RAW 264.7 cells. Cells were stimulated with RANKL/M-CSF and Intracellular ROS production was detected by CM-DCF fluorescence...

After verifying ROS generation within cells during RANKL-stimulated osteoclast differentiation, the relationship between H2O2 and various downstream signaling pathways of osteoclast differentiation such as IκBα, AKT, JNK, p38 and ERK were evaluated without RANKL stimulation. In western blot analysis, IκBα and AKT activation was induced in a dose-dependent manner by exogenous treatment with H2O2 (Figure 2C). The phosphorylation of IκBα by degradation of IκBα and AKT signal activity were all observed to reach their maximum levels when cells were exposed to a dose of 500 uM H2O2. Additionally, MAPK signals activity was detected in H2O2 treated cells (Figure 2D), and the phosphorylation of ERK, JNK, and p38 increased significantly with exposure to H2O2 in a dose-dependent manner.

To assess the antioxidant effect of simvastatin, we measured the scavenging activity of simvastatin against exogenous H2O2 at different concentrations with Amplex Red reagent. As shown in Figure 3A, content of the exogenous H2O2 45 µM concentrations significantly decreased when 50 nM of simvastatin was added. However, simvastatin had little antioxidant effect on H2O2 when used at a concentration of 30 µM. This indicates a dose-dependent antioxidant effect of simvastatin against H2O2 at concentrations of 45 µM or higher. Therefore, our results confirm that simvastatin acts as an antioxidant above some H2O2 concentrations.

Inhibition of ROS by simvastatin following exogenous H2O2 stimulation and RANKL stimulation in osteoclast differentiation. The amount of H2O2 was measured by scavenging activity of simvastatin against exogenous H2O2 at different concentrations with Amplex...

After verifying the effect of simvastatin against exogenous H2O2, the ability of simvastatin to scavenge intracellular ROS was investigated on cells undergoing RANKL-stimulated osteoclast differentiation. There was a marked decrease in the intensity of DCF fluorescence, a ROS indicator, within these cells. This was found to occur in a dose-dependent manner in simvastatin treated cell cultures during RANKL-induced osteoclast differentiation (Figure 3B). Additionally, an Amplex Red hydrogen peroxide Assay was performed in order to quantify the inhibitory effect of simvastatin on ROS generation during RANKL-induced osteoclast differentiation (Figure 3C). As opposed to the gradual increase of H2O2 amount within RANKL-stimulated cells for 60 min, the amount of H2O2 decreased significantly in a dose-dependent manner during RANKL-induced osteoclast differentiation when the cells were treated with simvastatin. The highest inhibitory effect of simvastatin on ROS generation was observed at a dose of 50 nM simvastatin.

In order to elucidate the underlying mechanism of simvastatin's inhibitory effect on osteoclast differentiation through ROS degeneration, we investigated the activation/inactivation of multiple downstream signaling molecules such as MAPKs, AKT and IκBα. These molecules are known to participate in osteoclast differentiation pathways during RANKL-stimulated osteoclast differentiation. RAW 264.7 cells were stimulated with RANKL in either the presence (50 nM concentration) or absence of simvastatin. Each signaling activity was detected with specific phospho-antibodies of the respective molecule's active form. As shown in Figure 4A, the highest phosphorylation of IκBα was detected after 15 min of RANKL treatment. However, co-incubation with simvastatin reduced such RANKL-induced phosphorylation of IκBα. In addition, RANKL-induced AKT activation was also blocked by simvastatin. Similarly, RANKL-induced activations of ERK, JNK and p38 were detectable within 15 min, but simvastatin reduced these RANKL-induced MAPKs signal activities (Figure 4B).

Effects of simvastatin on RANKL-induced p-IκBα, p-AKT, MAPKs activation in osteoclasts. RAW 264.7 cells were pretreated with simvastatin for 30 min, and then stimulated with RANKL for the indicated times. Whole cell lysates were immunoblotted...

Discussion

Bone resorption is a process of bone remodeling which is known to be mediated by osteoclasts, which result in bone loss by eliminating the bone's mineralized matrix. It has been well reported that excessive bone resorption can be related to bone loss diseases such as osteoporosis and rheumatoid arthritis (Gough et al., 1994). To date, various drugs have been developed and applied to treat such bone loss related diseases. Simvastatin is known to be one of these drugs used to suppress bone resorption by inhibiting osteoclastogenesis (Yamashita et al., 2010). However, the underlying biological mechanism of simvastatin's inhibitory effect on osteoclast differentiation has yet to be elucidated. Therefore, we studied the inhibitory effect of simvastatin on osteoclast differentiation and its underlying mechanism.

In this study, simvastatin-mediated inhibition of RANKL-induced osteoclast differentiation was proven. This was shown by a dose-dependent decrease of the formation of TRAP positive cells, as well as a dose-dependent down-regulation of TRAP gene expression. Simvastatin prevents the production of ROS and this action attenuates the H2O2-induced early signaling activities including AKT, JNK, p38, ERK and NF-κB signaling pathways, which subsequently suppresses osteoclast formation.

It is well known that low concentrations of ROS mediated signals are required for the activation of downstream signaling pathways (Rhee, 1999; Dröge, 2002) and the generation of ROS is controlled by the binding of various cytokines to receptors (Bonizzi et al., 1999). In the osteoclast differentiation pathway, RANKL binds to the RANK receptor and then stimulates intracellular ROS generation. This ROS is known to stimulate osteoclast differentiation and participate in early signaling events associated with osteoclast activation for bone resorption (Hall et al., 1995). Here, we show that ROS was generated within RAW 264.7 cells during RANKL/M-CSF-simulated osteoclast differentiation. This ROS production during RANKL-simulated osteoclast differentiation decreased distinctly when the RAW 264.7 cells were co-treated with simvastatin. Previous studies have reported on the inhibitory function of simvastatin on osteoclast differentiation by blocking the RANKL-induced signaling pathway (Ahn et al., 2008; Yamashita et al., 2010). It has also been reported that simvastatin has antioxidant activity (Giroux et al., 1993; Girona et al., 1999; Mason, 1999; Tomás et al., 2000; Davignon et al., 2004). From these findings, we hypothesized that simvastatin might suppress osteoclast differentiation by scavenging the generated intracellular ROS which acts as a secondary messenger in the RANKL osteoclast differentiation signaling pathway.

Previous studies have shown that simvastatin inhibited RANKL-induced NF-κB pathway activation through the suppression of IκBα phosphorylation, IκBα degradation, and IκBα kinase activity (Ahn et al., 2008). This study confirmed these results that simvastatin down-regulated IκBα phosphorylation and IκBα degradation both of which are known to participate in the RANKL-induced osteoclast differentiation signaling pathway. In this study, the main intracellular signal pathways engaged by RANK were also investigated in order to elucidate the underlying mechanisms of simvastatin's inhibitory effects on this pathway. It has been previously found that RANKL-induced osteoclast differentiation is mediated by RANK. RANK mainly activates NF-κB, AKT, JNK, P38 and ERK signaling pathways (Kim et al., 2009; Hasegawa et al., 2010; Lee et al., 2010). Other drugs have been found to suppress these RANKL-induced signaling pathways. Curcumin, a natural polyphenolic derogate extracted from turmeric, has also been found to suppress osteoclast differentiation and osteoclast function. These effects were accompanied by the inhibition of IκBα phosphorylation and NF-κB activation (Von Metzler et al., 2009). Luteolin has also been found to inhibit osteoclasts, however, this does not affect RANKL-induced MAPKs expression and activation. Instead, this antioxidant was found to affect the phosphorylation of ATF2, which is downstream of the p38 MAPK signaling pathway (Lee et al., 2009). Sauchinone also attenuates RANKL-induced NF-κB, ERK and p38 MAPKs activation in osteoclast differentiation. However, the activation of JNK by RANKL was not affected by sauchinone (Han et al., 2007). In our study, we also detected inhibition effects of RANKL-induced signaling pathways by simvastatin. Simvastatin inhibited not only IκBα signal but also the AKT, and MAPKs such as JNK, p38 and ERK signaling pathways which are normally activated through ROS in osteoclast differentiation. Simvastatin strongly attenuates RANKL-induced AKT and JNK signaling.

In conclusion, we analyzed the effect of osteoclast differentiation by simvastatin and identified one mechanism by which simvastatin acts as an inhibitor. These results show that simvastatin suppresses RANKL-induced osteoclastogenesis through inhibition of ROS-induced signaling pathways. This study elucidated the mechanism by which simvastatin acts as an osteoclast inhibitor by blocking ROS and help contribute to further clarification of the mechanisms underlying osteoporosis and other pathological bone-loss diseases. Also, antioxidants may have therapeutic implications for inhibiting bone resorption.

Cell culture and differentiation into osteoclasts

RAW 264.7 (mouse macrophage) cells were obtained from the Korean Cell Lines Bank (KCLB, Seoul, Korea) and were cultured in Dulbecco's modified Eagles medium (DMEM, GIBCO, NY) supplemented with 10% fetal bovine serum (FBS, GIBCO) and 1% antibiotic-antimycotic in a humidified 5% CO2 atmosphere. The cells were incubated in the presence of RANKL (200 ng/ml) and M-CSF (40 ng/ml) with or without varying doses of simvastatin for 7 days. The culture medium was replaced every 3 days.

Tartrate-resistant acid phosphatase (TRAP) staining

Cells were fixed by soaking in 3.7% formaldehyde for 20 min and were then placed in 0.1% triton X-100 for 10 min. Following this, the cells were stained using the Leukocyte Acid Phosphatase Assay kit following the manufacturer's instruction. TRAP-positive multinucleated cells containing three or more nuclei were considered to be osteoclasts.

RT-PCR and real-time quantitative PCR

Total RNA was purified using an RNeasy Plus Mini Kit (Qiagen, CA) and cDNA was synthesized from 1 µg of total RNA using AccuPower® RT PreMix (Bioneer, Daejeon, Korea). All PCR amplifications were performed using AccuPower® PCR PreMix (Bioneer). The primers for TRAP were 5'-ACT TCC CCA GCC CTT ACT ACC G-3' (sense) and 5'-TCA GCA CAT AGC CCA CAC CG-3' (antisense), and the primers for GAPDH were 5'-ACT TTG TCA AGC TCA TTT CC -3' (sense) and 5'-TGC AGC GAA CTT TAT TGA TG -3' (antisense). To confirm RT-PCR results in this study, the same cDNA samples were analyzed using real-time PCR. Primers were selected using the same primers as used in RT-PCR. For relative quantitation, the reactions were tested using DyNAmo™ SYBR® Green qPCR Kit (Finnzymes, Espoo, Finland) and PCR processing was carried out in an iCycler (Bio-Rad, Hercules, CA). The copy number of the target gene was normalized to an endogenous reference, GAPDH. The fold change from normal samples was set at 1-fold and the ratio of the normalized fold change was calculated.

Reactive oxygen species (ROS) assay

Intracellular ROS images were measured using the fluorescence of DCF, which is the oxidized product of 2',7'-dichlorofluorescein diacetate (DCFH) in stained cells. Pre-osteoclasts were stimulated with only RANKL and M-CSF or with these signal molecules combined with simvastatin. Afterwards, the cells were washed in Hank's balanced salt solution (HBSS) and then incubated in the dark for 30 min with 10 µM DCFH-DA. The fluorescence of DCF was detected by confocal microscopy. Also, the amount of H2O2 in each lysate was measured by using an Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes). In the presence of peroxidase, the Amplex Red reagent reacted with H2O2 to produce a red-fluorescent oxidation product and this process was performed per manufacturer's instruction.

Western blotting

Cells were lysed by the addition of cold RIPA lysis buffer containing 0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA along with protease and phosphatase inhibitors. An equal amount of protein (30 µg) was subjected to SDS-PAGE and transferred to a nitrocellulose transfer membrane. After blocking with 5% skim milk, the membrane was probed with anti-phospho JNK, phospho ERK, phospho p38, phospho AKT, phospho IκBα followed by incubation with an appropriate secondary antibody conjugated to horseradish peroxidase. The membranes were then stripped and reprobed with anti-JNK, ERK, p38, AKT, IκBα and β-actin. Signals were detected using a ChemiDoc XRS System (Bio-Rad).

Statistical analysis

Each experiment was performed at least three times. All quantitative data are expressed as mean ± S.D. Statistical differences were analyzed by Student's t-test (*P < 0.01).

Acknowledgements

This Research was supported by the Program of Kyung Hee University for the Young Researcher of Medical Science in 2007 (KHU-20071500).